Biological thiols play a major role in regulating various biological events such as redox-methyl transfer and CoA-related reactions. The concentration levels of intracellular thiols change dramatically in response to oxidative stress, which has been associated with a number of diseases, including cancer, AIDS, Alzheimer's and cardiovascular disease. Therefore, the rapid, highly sensitive, and selective determination of intracellular thiols is of great importance for investigating its role in cellular functions and disease detection and diagnosis. Among the various analytical techniques employed today, optical detection has proven to be the most convenient methods of all and fluorescence is an important optical detection method due to its high sensitivity, low detection limit and have an advantage of intracellular detection.
Fluorescence based thiol sensors owing to the nucleophilicity of thiols undergo Michael addition, cyclisation with aldehyde, cleavage of sulphonamide, sulfonate ester and disulphide. Squaraine dyes have been extensively investigated as chemosensors and chemodosimeters.
Protein labelling is an important technique that enables the direct visualization of many biological events such as protein functions, protein dynamics, protein—protein interactions and for the detection of various biologically relevant analytes. Labelling strategies with small organic fluorophore have attracted much attention. Depending on the nature of interaction between the receptor protein and the ligand, the labelling can be either high affinity noncovalent or site specific covalent labelling. Herein we report a novel fluorescent molecule that switches the mode of interaction between covalent and noncovalent labelling giving two different spectral responses by changing the pH of the solution and detect the serum albumin protein selectively among other thiol containing competing molecules.
Reference may be made to a symmetrical squaraine based fluorescent probe that detect thiols at physiological pH window with high sensitivity reported by R. M. Manez et al. J. Am. Chem. Soc. 2004, 126, 4064. The main drawback is the detection of thiol is based on fluorescence quenching and hence it is not applicable for the detection of intracellular thiol imaging.
Another reference may be made to use a symmetrical squaraine dye that detects thiols ratiometrically reported by A. Ajayaghosh et al. Angew. Chem. Int. Ed. 2008, 120, 8001. The main drawback is that the reactivity of the probe is very poor and the detection of thiol occurs at basic pH, 9.6 which we cannot use for the detection of thiol content in cells due to the lack of biocompatibility.
References may be made to fluorescent molecule that detects thiols inside cells which were pre-treated with a thiol deactivating molecule, NEM of concentration 0.2-1 mM reported by S Kim et al. Org Lett. 2011, 13, 6. S. Kim et al. Chem. Commun., 2011, 47, 5142. The main drawback is the detection of thiols inside the cells is limited to only in milli molar range.
However, as evident from the above references a fluorescent probe that could image thiol fluctuations inside cells which were pre-treated with NEM of concentrations from milli molar to micro molar range is still unknown.
References may be made to a protein labeling method, termed as ligand-directed tosyl (LDT) chemistry, which can site-specifically introduce a synthetic probe to a protein with the concomitant release of the affinity ligand. The molecule initially forms a complex with protein and then under goes covalent labeling as reported by I. Hamachi. Nat. Chem. Biol. 2009, 5, 341. The major drawback is that the spectroscopic response remains the same for both modes of interactions and the initial complex formed is less stable and immediately gives rise to a reaction that allows only covalent labeling, and hence called as affinity based irreversible covalent labeling.
As evident from the above reference, the fluorescent probe in the present invention specifically switches the mode of interaction with proteins between a noncovalent and covalent labeling, which can be controlled with an external stimulus resulting in distinct signal response is a new and novel approach.
Reference may be made to fluorescent pH nanosensor by incorporating two different fluorophores to carbon nanodots. By changing the ratio of these fluorophores functionalised on the surface of carbon nanodots the region of sensitivity in the pH scale can be tuned as reported by H. Ma et al. Angew. Chem. Int. Ed. 2012, 51, 1. The drawback of this work is, tuning based on ratio of fluorophores functionalised over carbon nanodots is possible through fresh synthesis of carbon nanodots with fluorophores in different ratios.
Another reference may be made to use fluorescent dyes with polymers that form nanoparticles. Different dyes shows sensitivity in different regions of the pH scale as reported by J. Gao et al. J. Am. Chem. Soc. 2012, 134, 7803. The main drawback of this work is that for different regions of sensitivity, polymers with different dyes have to be synthesised separately.
Since the labeling process is reversible with respect to the variation of pH of the solution, we employed the dye-protein complex for monitoring pH variations inside the cells. Intracellular pH plays an important role in regulating various cellular events including cell growth, receptor mediated signal transduction, enzymatic activity, calcium regulation and cell adhesion. Under normal physiological conditions the pH is maintained within a range of 7.35-7.45. Small deviation out of this range can cause cardiopulmonary and neurologic problems (e.g., Alzheimer's disease) and more extreme variations can be fatal. So detection of pH changes inside the cells is of great importance. Small molecular fluorescent probes as well as fluorescent proteins have been widely used for intracellular pH detection. Herein we use the dye protein complex in a particular ratio to monitor minor pH fluctuations inside the cells
As evident from the above references, fluorescence based pH sensors either detect the pH changes within a narrow pH window with high sensitivity or detect pH in a broad range with less sensitivity. However a simple probe that can be used to detect pH variations in a broad region with high sensitivity is still not known. Herein we report the dye-protein complexes of various ratios that was prepared just by mixing in different ratios and can be used to detect pH from 4.6-11.6 with high sensitivity.
Squaraine dyes have been used as a colorimetric and fluorescent probe for the detection of thiols. The first report came from R. M. Manez et al. J. Am. Chem. Soc. 2004, 126, 4064. In this work the authors have used a symmetrical squaraine dye of aniline derivative for the detection of thiol at physiological (6.5-7.5) pH window with high sensitivity. Since the detection of thiol is basically based on fluorescence quenching, this probe shall not be useful for cell imaging. Later we have reported a symmetrical squaraine dye with two chromophores on both side of the ring which can be activated upon interaction of the thiols (Ajayaghosh et al. Angew. Chem. Int. Ed 2008, 120, 8001). Due to the poor reactivity and detection of thiols occur at basic pH window (9.6), the dye was not biocompatible and not useful for cell imaging purposes.
In order to obviate the drawbacks associated with the known squarine dyes there is a need to provide the squarine based fluorescent probes. Accordingly, we have synthesized an unsymmetrical squaraine dye that detects thiols by “turn-on” fluorescence with high sensitivity at physiological (7.2-7.8) pH. This high reactivity at physiological pH window and fluorescence “turn-on” response toward thiols helps the inventors to use this probe for the detection of thiol fluctuations inside the biological cells from milli molar to micro molar ranges.